Mycobacterium tuberculosis is the most deadly bacterial pathogen in the world, killing 1.2 million people yearly and infecting over 8 million (WHO, 2013). Although chemotherapy against TB exists, a rapid global increase of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) cases makes new drugs with novel killing mechanisms an urgent need. A major drawback of current TB chemotherapy is its long duration, which increases the probability of relapse and the emergence of drug resistance. The underlying problem of this phenomenon is a population of non-replicating, drug-tolerant bacilli, the so-called persisters. However, current TB drugs are mainly effective against replicating and metabolically active bacteria. Preferably, new drugs kill fast (within weeks) and target actively growing as well as persister cells. Using a multidisciplinary approach, we have identified a novel drug target in methionine biosynthesis of M. tuberculosis. Our preliminary results are very promising, as they show rapid in vitro sterilization of a M. tuberculosis methionine auxotroph as well as complete lack of virulence in immunocompetent and immunocompromised mice. This is intriguing because most available TB antibiotics do not rapidly sterilize cultures. Metabolomics and transcriptomic analysis revealed a systemic metabolic shutdown by an unprecedented multi-target inhibition mechanism. The prospect of killing M. tuberculosis by causing rapid biosynthetic and metabolic seizure is very attractive for drug discovery. The goal of this proposal is to validate this drug target in vitro and in vivo and o develop a reporter strain for whole cell inhibitor screening. This will set the stage for a comprehensive high-throughput inhibitor screen against this target in the near future.